Distinct Phosphorylation Sites Differentially Influence Facilitated Transport of an NLS-Protein End Its Subsequent Intranuclear Binding

We measured the nuclear transport of radiolabeled fusion proteins consisting of variants of the Simian Virus 40 large T antigen′s nuclear localization sequence region linked to β-galactosidase, itself a cytoplasmic protein. We microinjected the fusion protein variants into the cytoplasm of living Xenopus oocytes or supplied them to the surface of oil-isolated oocyte nuclei via paired beads or cytoplasm. Presence of the cdc2 kinase site (124T) on the amino flank of the nuclear localization sequence (126PKKKRKV132) greatly enhances facilitated transport through the nuclear pore complex; additional presence of the casein kinase II site (112S) enhances subsequent intranuclear binding.